Eastern equine encephalitis virus (EEEV), an emerging mosquito-borne virus, causes the deadliest arboviral disease in the U.S., with case-fatality rates up to 75%. However, EEEV also circulates throughout South and Central America (SA), where many people are exposed to infected mosquitoes yet with little evidence of disease. Epidemiologic and pathogenesis studies suggest that SA strains are poorly infectious for people, possibly related to higher sensitivity to type I interferon (IFN) and greater IFN induction following infection of myeloid cells. The major contrast in EEE incidence in NA vs. SA changed dramatically in 2010, when the first human outbreak ever documented in Latin America occurred in Panama (PA). Clinical disease was similar to that seen in NA, and our genetic and epidemiologic data indicate that an enzootic PA EEEV strain increased its infectivity and/or virulence since 1986. Because SA EEEV vectors bite people more frequently than NA vectors, this change in EEEV virulence could have major public health implications if the 2010 PA strain spreads to other parts of Latin America, or if similar viral changes occur in the region. Understanding the genetic and phenotypic changes in the 2010 PA strain is therefore critical to our long-term goal of reducing arboviral disease. Recently, our reverse genetic approaches combined with experimental infections of animal and in vitro models led to major advances in understanding arboviral disease emergence, including Venezuelan equine encephalitis and chikungunya. However, to apply these approaches to gain a mechanistic understanding the 2010 PA EEE emergence, better in vitro and small animal EEE models are needed. The lack of such models currently represents the major bottleneck to a better understanding of human EEE and developing improved interventions. In this exploratory project, we will exploit recent, major advances in the understanding of EEE molecular pathogenesis to develop models that predict differential human virulence of 1984 vs. 2010 PA EEEV strains using 2 specific aims: 1. Test cotton rats, and if needed, a variety of mouse strains and hamsters for their ability to reproduce human-like disease and to mirror the typical differences in infectivity/virulence between NA and previously isolated SA EEEV strains, and to compare 1984 and 2010 PA strains in these models. 2. Perform comparative in vitro molecular pathogenesis studies of PA and representative NA and SA EEEV strains to identify markers of human virulence. These models will allow us to test the hypothesis that genetic changes in PA EEEV strains between 1986-2010, including in the 3' untranslated genome region, altered the virus' infectivity for dendritic cells and the induction of interferon responses, resulting in greater virulence and infectivity for humans. This developmental project will advance the mechanistic understanding not only of EEEV emergence, but also of alphavirus pathogenesis, which will lead to improved surveillance, diagnostics, treatment and vaccine development.